title: Resolution by unassisted Top3 points to template switch recombination intermediates during DNA replication.
abstract: The evolutionarily conserved Sgs1/Top3/Rmi1 (STR) complex plays vital roles in DNA replication and repair. One crucial activity of the complex is dissolution of toxic X-shaped recombination intermediates that accumulate during replication of damaged DNA. However, despite several years of study the nature of these X-shaped molecules remains debated. Here we use genetic approaches and two-dimensional gel electrophoresis of genomic DNA to show that Top3, unassisted by Sgs1 and Rmi1, has modest capacities to provide resistance to MMS and to resolve recombination-dependent X-shaped molecules. The X-shaped molecules have structural properties consistent with hemicatenane-related template switch recombination intermediates (Rec-Xs) but not Holliday junction (HJ) intermediates. Consistent with these findings, we demonstrate that purified Top3 can resolve a synthetic Rec-X but not a synthetic double HJ in vitro. We also find that unassisted Top3 does not affect crossing over during double strand break repair, which is known to involve double HJ intermediates, confirming that unassisted Top3 activities are restricted to substrates that are distinct from HJs. These data help illuminate the nature of the X-shaped molecules that accumulate during replication of damaged DNA templates, and also clarify the roles played by Top3 and the STR complex as a whole during the resolution of replication-associated recombination intermediates.
authors.affiliation: From the Department of Pathology and Laboratory Medicine.
authors.forename: M Rebecca
authors.initials: MR
authors.lastname: Glineburg
authors.forename: Alejandro
authors.initials: A
authors.lastname: Chavez
authors.forename: Vishesh
authors.initials: V
authors.lastname: Agrawal
authors.forename: Steven J
authors.initials: SJ
authors.lastname: Brill
authors.forename: F Brad
authors.initials: FB
authors.lastname: Johnson
mesh_terms: D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair / Q000502:physiology; D004261:DNA Replication / Q000502:physiology*; D045566:DNA, Cruciform / Q000235:genetics / Q000378:metabolism; D004271:DNA, Fungal / Q000096:biosynthesis* / Q000235:genetics; D043326:Glucan 1,3-beta-Glucosidase / Q000235:genetics / Q000378:metabolism; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic / Q000502:physiology*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D045566:DNA, Cruciform; D004271:DNA, Fungal; D046912:Multiprotein Complexes; D029701:Saccharomyces cerevisiae Proteins; C502374:TOP3 protein, S cerevisiae; D043326:Glucan 1,3-beta-Glucosidase; C117648:beta-1,3-exoglucanase
keywords: DNA Damage; DNA Replication; DNA Topoisomerase; DNA Topology; Holliday Junction; Homologous Recombination; Rec-X; Sgs1; Template Switch Recombination; Top3